The present invention relates to an enzyme xcex1-1,4-glucan lyase (xe2x80x9cGLxe2x80x9d) and, in particular, its use to prepare 1,5-D-anhydrofructose (xe2x80x9cAFxe2x80x9d) from substrates based on xcex1-1,4-glucan.
The present invention also relates to the use of a sugar, in particular 1,5-D-anhydrofructose (xe2x80x9cAFxe2x80x9d) prepared by the method of the present invention, as an anti-oxidant, in particular as an anti-oxidant for food stuffs and beverages.
The present invention relates to the use of 1,5-D-anhydrofructose (xe2x80x9cAFxe2x80x9d), in particular AF prepared by the method of the present invention, as a sweetener, in particular as a sweetener for foodstuffs and beverages, preferably human foodstuffs and beverages.
FR-A-2617502 and Baute et al in Phytochemistry [1988] vol. 27 No.11 pp3401-3403 report on the production of AF in Morchella vulgaris by an apparent enzymatic reaction. The yield of production of AF is quite low. Despite a reference to a possible enzymatic reaction, neither of these two documents presents any amino acid sequence data for any enzyme let alone any nucleotide sequence information. These documents say that AF can be a precursor for the preparation of the antibiotic pyrone microthecin.
Yu et al in Biochimica et Biophysica Acta [1993] vol 1156 pp313-320 report on the preparation of GL from red seaweed and its use to degrade xcex1-1,4-glucan to produce AF. The yield of production of AF is quite low. Despite a reference to the enzyme GL this document does not present any amino acid sequence data for that enzyme let alone any nucleotide sequence information coding for the same.
A typical xcex1-1,4-glucan based substrate is starch. Today, starches have found wide uses in industry mainly because they are cheap raw materials.
Starch degrading enzymes can be grouped into various categories. The starch hydrolases produce glucose or glucose-oligomers. A second group of starch degrading enzymes are phosphorylases that produce glucose-1-phosphate from starch in the presence of inorganic phosphate.
AF has also been chemically synthesisedxe2x80x94see the work of Lichtenthaler in Tetrahedron Letters Vol 21 pp 1429-1432. However, this chemical synthesis involves a large number of steps and does not yield large quantities of AF.
The chemical synthetic route for producing AF is therefore very expensive.
There is therefore a need for a process that can prepare AF in a cheap and easy manner and also in a way that enables large quantities of AF to be made.
Furthermore, anti-oxidants are typically used to prevent oxygen having any deleterious effect on a substance such as a foodstuff. Two commonly used anti-oxidants are GRINDOX 142 (antioxidant) and GRINDOX 1029 (antioxidant). These anti-oxidants contain many components and are quite expensive to make.
There is therefore a need to have a simpler and cheaper form of anti-oxidant.
Furthermore, sweeteners are often used in the preparation of foodstuffs and beverages. However, many sweeteners are expensive and complex to prepare.
There is therefore a need to have a simpler and cheaper form of sweetener.
According to a first aspect of the present invention there is provided an enzyme comprising at least any one of the amino acid sequences shown as SEQ. ID. No.s 3-4, or any variant thereof.
According to a second aspect of the present invention there is provided a nucleotide sequence coding for the enzyme of the first aspect of the present invention.
Preferably the nucleotide sequence is a DNA sequence.
According to a third aspect of the present invention there is provided a nucleotide sequence comprising a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of SEQ. ID. No. 1 or SEQ. ID. No. 2.
According to a fourth aspect of the present invention there is provided a method of preparing the sugar 1,5-D-anhydrofructose comprising treating an xcex1-1,4-glucan with the enzyme xcex1-1,4-glucan lyase, characterised in that enzyme is used in substantially pure form and wherein the enzyme is isolated from algae alone.
According to a fifth aspect of the present invention there is provided a method of preparing the sugar 1,5-D-anhydrofructose comprising treating an xcex1-1,4-glucan with the enzyme xcex1-1,4-glucan lyase characterised in that enzyme comprises at least any one of the amino acid sequences shown as SEQ. ID. No.s 3-4, or any variant thereof.
According to a sixth aspect of the present invention there is provided the sugar 1,5-D-anhydrofructose when prepared by the method of the present invention.
According to a seventh aspect of the present invention there is provided the use of a reagent that can increase the hydrophobicity of the reaction medium to increase the stability and activity of the GL enzyme.
According to an eighth aspect of the present invention there is provided the use of AF prepared by the method of the present invention as an anti-oxidant.
According to a ninth aspect of the present invention there is provided the use of AF prepared by the method of the present invention as a sweetener.
Preferably the enzyme is obtainable from algae, preferably it is obtainable from Gracilariopsis lemaneiformis. 
Preferably the enzyme comprises at least any one of the amino acid sequences shown as SEQ. ID. No.s 3-4, or any variant thereof.
Preferably the enzyme is obtained from the expression of a nucleotide sequence coding for the enzyme.
Preferably the nucleotide sequence is a DNA sequence.
Preferably the DNA sequence comprises a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of SEQ. ID. No. 1 or SEQ. ID. No. 2.
Preferably if the glucan contains links other than and in addition to the xcex1-1,4- links the xcex1-1,4-glucan lyase is used in conjunction with a suitable reagent that can break the other links.
Preferably the glucan is starch.
Preferably a glucanohydrolase is used in conjunction with the xcex1-1,4-glucan lyase.
Preferably the starch is used in high concentrationxe2x80x94such as up to about 25% solution.
Preferably the hydrolase is at least one of pullanase or isoamylase.
Preferably the xcex1-1,4-glucan lyase is bound to a support or, more preferably, is in a dissolved form.
Preferably the enzyme is isolated and/or further purified from algae alone using a gel that is not degraded by the enzyme.
Preferably the gel is based on dextrin or derivatives thereof, preferably the gel is a cyclodextrinxe2x80x94more preferably xcex2-cyclodextrin.
Preferably the substrate is treated with the enzyme in the presence of a buffer.
Alternatively, preferably the substrate is treated with the enzyme in the presence of at least substantially pure water.
Preferably the substrate is treated with the enzyme in the absence of a co-factor.
Preferably the enzyme is used in combination with amylopectin or dextrin.